Neissena gonorrhoeae causes both localized uncomplicated infections at mucosal surfaces and more invasive forms of disease including pelvic inflammatory disease and disseminated gonococcal infection. The ability of gonococci to evade the antimicrobial action of mediators of innate host defense at mucosal surfaces or in the bloodstream is of likely importance in its ability to survive eariy during infection. In this project we will focus on determining whether mechanisms of gonococcal resistance to antimicrobial agents of innate host defense impact infectivity and/or in vivo fitness. In Specific Aim 1 we will test the role of the MtrC-MtrD MtrE efflux pump in determining the ability of gonococci to Infect the male urethra. To our knowledge, this will be the first test of the importance of any microbial efflux pump in promoting an infection in humans. The MtrC-MtrD-MtrE efflux pump endows gonococci with a mechanism to export hydrophobic antibiotics and host-derived antimicrobials (antimicrobial peptides, bile salts and progesterone) that bathe mucosal surfaces. The proteins that fomn the pump are encoded by the mtrCDE operon, which is transcriptionally regulated by cis- and frans-acting regulatory control processes. These regulatory systems modulate levels of gonococcal fitness in a murine model of lower genital tract infection and we will now test if they also modulate in vivo fitness in humans. In Specific Aim 2 we will determine whether mutations that alter lipooligosaccharide structure and are known to result in changes in gonococcal susceptibility to the killing action of normal human serum or cationic antimicrobial peptides impact gonococcal infectivity and/or in vivo fitness. We hypothesize that mediators of the innate host defense system function at the genital mucosal surface as antibiotics and as a consequence of their antimicrobial action against gonococci, resistant variants ultimately emerge and these have a competitive advantage during infection. Accordingly, in Specific Aim 3 we will isolate laboratory-derived mutants of gonococci with altered levels of susceptibility to host-derived antimicrobials and test if such strains have increased or decreased fitness in vivo. The results will provide important insights regarding the ability of gonococci to adapt to the in vivo environment.